Proliferative growth of normal cells requires an orderly progression through a series of distinct steps, a process known as the cell cycle (Alberts et al., Cell Growth and Division, Garland Publishing, Inc., New York). Progression through the cell cycle is modulated by nutrient availability, cell size, and growth factors through complex signaling pathways involving phosphorylation cascades and the strictly regulated expression and stability of specific proteins required at each phase of the cell cycle. In addition, the sequence of cell cycle events is rigorously controlled at specific checkpoints to ensure that each discrete stage in the cell cycle has been completed before the next is initiated. Human diseases associated with abnormal cell proliferation result when these rigorous controls on cell cycle progression are perturbed.
The invention relates to novel genes which function in cell cycle regulation. In a particular embodiment, the genes are derived from vertebrates, including mammalian cells, particularly those derived from Xenopus or human cells, and function in the regulation of DNA replication and/or entry of a cell into mitosis. In one embodiment, the gene is a human gene called Hscdc6 and in another embodiment the gene is a Xenopus gene called Xcdc6. In one embodiment, the genes have a DNA sequence comprising at least one DNA sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and combinations thereof; the invention also pertains to the complementary DNA sequences thereof. The present invention also relates to genes which function in the regulation of DNA replication or the entry of a cell into mitosis and which have a nucleotide sequence which hybridizes under conditions of medium stringency to at least one DNA sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 and 26.
In particular embodiments, the isolated nucleic acid molecule encodes a protein or polypeptide with the same amino acid sequence as the endogenous protein or polypeptide. In another embodiment, the isolated nucleic acid molecule has the same nucleotide sequence as the endogenous gene encoding the protein or polypeptide.
Accordingly, this invention pertains to an isolated Hscdc6 gene or an Xcdc6 gene, or an active derivative or fragment thereof. The isolated gene is characterized by its ability to regulate the cell cycle as described herein. In particular embodiments, the expressed protein or polypeptide is purified to homogeneity or is substantially free of other proteins (i.e., isolated).
The invention also pertains to novel gene products, e.g., polypeptides or proteins, encoded by the vertebrate genics described herein, or an active derivative or fragment thereof. In a particular embodiment, the polypeptide or protein has the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In another embodiment, the gene product is a recombinant human or Xenopus polypeptide or protein which regulates DNA replication and/or the entry of a cell into mitosis. In one embodiment, the encoded protein or polypeptide is a fragment having DNA replication regulation and/or mitosis regulation activity. In another embodiment, the encoded protein or polypeptide is a derivative possessing substantial sequence identity with the endogenous protein or polypeptide.
The invention also relates to DNA constructs comprising the nucleic acid molecules described above operatively linked to a regulatory sequence, and to recombinant host cells, such as bacterial cells, fungal cells, plant cells, insect cells and mammalian cells, comprising the nucleic acid molecules described above operatively linked to a regulatory sequence.
The invention also pertains to an antibody, or an antigen-binding fragment thereof, which selectively binds to the described protein or polypeptide, or an active derivative or fragment thereof. One embodiment of the invention relates to monoclonal antibodies having specificity for Hscdc6. In particular, the invention encompasses the hCdc6-26, hCdc6-37, hCdc6-34, hCdc6-39, and hCdc6-41 monoclonal antibodies or an immunoglobulin antigen binding region or fragment derived from the hCdc6-26, hCdc6-37, hCdc6-34, hCdc6-39, and hCdc6-41 monoclonal antibodies.
Another embodiment of the claimed invention relates to the monoclonal antibodies derived from the hybridoma deposited with the American Type Culture Collection (ATCC), Accession Numbers: HB-12590 and HB-12591 Oct. 30, 1998, as well as to the deposited hybridomas themselves. Additionally, the invention relates to a humanized or chimeric immunoglobulin having specificity for Hscdc6 comprising an antigen binding region of non-human origin (e.g., the complementarity determining region (CDR) that is derived from the hCdc6-26, hCdc6-37, hCdc6-34, hCdc6-39, or hCdc6-41 monoclonal antibody). The humanized or chimeric immunoglobulin can further comprise at least a portion of human origin (e.g. a human constant region and/or a human framework region (FR)).
The invention also relates to a method for assaying the presence of the described protein or polypeptide in a cell, e.g., in a sample from an individual, comprising contacting said cell with an antibody which specifically binds to the protein or polypeptide. Embodiments of the claimed invention include Enzyme-Linked Immnosorbent Assay (ELISA), competition ELISA assays, RadioImmuno-Assays (RIA), immunofluorescence and immunohistochemical assays which involve assaying Hscdc6 in a sample using the monoclonal antibodies having specificity for Hscdc6, as described herein. The invention involves determining the presence or absence of Hscdc6 comprising combining the sample to be tested with an antibody (e.g., hCdc6-26, hCdc6-37, hCdc6-34, hCdc6-39, or hCdc6-4 1) having specificity for Hscdc6, and then detecting or measuring the formation of the complex between the antibody and the antigen. The antibodies are detectably labeled (e.g., radioactive, fluorescently, biotinylated or HRP-conjugated) to facilitate detection of the complex.
The claimed invention also pertains to methods for determining the presence or absence of a proliferative disorder (e.g., cancer) comprising determining the presence, absence, or the level of hscdc6, wherein the presence of hscdc6 or an elevated level of hscdc6, as compared to a control, standard, or baseline, indicates the presence of a proliferative disorder. An embodiment of the claimed invention includes determining the presence or absence of a proliferative disorder comprising determining the levels (e.g., presence or absence) of two or more markers for proliferative disease, wherein one of the markers is hscdc6. The additional marker can be a protein from the Mcm (mini-chromosome maintenance) family (e.g., Mcm-2, Mcm-3, Mcm-4, Mcm-5, Mcm-6, and Mcm-7). The invention also embodies methods for diagnosing or aiding in the diagnosis of a proliferative disease comprising determining the presence, absence or level of hscdc6, wherein the presence of hscdc6 or ail elevated level of hscdc6, as compared to a control, standard, or baseline, indicates a positive diagnosis for a proliferative disorder. These methods utilize the hscdc6 assays and/or monoclonal antibodies having specificity for hscdc6, as described herein.
Furthermore, the invention encompasses pharmaceutical compositions comprising the genes and proteins or polypeptides described herein, as well as methods of treating disease utilizing the compositions described herein. For example, the invention relates to a method of treating a tumor in an individual. In the method, an antagonist of Hscdc6 is administered to the individual, causing at least one of two possible results: inhibition of Hscdc6 function and inhibition of tumor cell DNA replication, with concomitant inhibition of tumor growth, or mitotic division of tumor cells with failure of DNA replication and tumor cell death. Such compositions comprise antibodies having a specificity for hscdc6(e.g,. hCdc6-26, hCdc6-37, hCdc6-39, hCdc6-34, and hCdc6-41).
The invention also relates to a method of treating a tumor in an individual comprising administering an agonist of Hscdc6 to the individual in such a manner that it enters tumor cells in the individual, introduction of the Hscdc6 agonist in G2 or M phase of the cell cycle prevents entry of the cell into mitosis, and thus results in tumor cell death. The invention also pertains to a method of inhibiting undesired cell proliferation in an individual comprising administering an agonist or antagonist of Hscdc6 to the individual in such a manner that the agonist or antagonist enters the cells in which it is desirable to inhibit proliferation.
An antagonist of Hscdc6 will prevent or reduce the activity of Hscdc6, and thereby prevent the replication of cellular DNA; cells with unreplicated DNA will enter mitosis and cell death will result. For example, methods of treatment include administering antagonists that are immunoglobulins having antigen binding regions of one of the hCdc6-26, hCdc6-37, hCdc6-34, hCdc6-39, or hCdc6-41 monoclonal antibodies. The method also includes administering, a humanized or chimeric immunoglobulin having antigen binding region or fragment derived from hCdc6-26, hCdc6-37, hCdc6-34, hCdc6-39, and hCdc6-41.
An agonist of Hscdc6 will prolong or increase the effects of Hscdc6, resulting in polyploidy and preventing mitosis, cells which are affected in this manner will undergo programmed cell death. The method of inhibiting cell proliferation can be used in the treatment of conditions associated with undesirable levels of cell proliferation, such as tumor growth or cancer.
The invention also encompasses a method of enhancing cell proliferation for therapy of a condition associated with loss of viable tissue in an individual comprising administering Hscdc6 or an agonist of Hscdc6 to an individual such that it enters cells in the individual. The activity of Hscdc6 or an Hscdc6 agonist causes initiation of DNA replication in the cell and entry of the cell into mitosis. The invention further relates to a method of diagnosing or aiding in the diagnosis of conditions associated with proliferative disorders in an individual; this method can also be used to predict the likelihood that an individual is at increased risk for a particular condition associated with abnormal cell proliferation. According to this method, by combining probes derived either from the isolated native sequence of the Hscdc6 gene or from the primers disclosed herein with DNA from an individual to be assessed, under conditions suitable for hybridization, it can be determined whether the individual possesses the gene. Hybridization conditions can be selected such that the probes will hybridize only with altered DNA and not with unaltered DNA; that is, the probes can be designed to recognize only particular alterations in the nucleic acid sequence of the gene, including addition of one or more nucleotides, deletion of one or more nucleotides or change in one or more nucleotides (including substitution of a nucleotide for one which is normally present in the sequence).
The claimed invention also pertains to kits (e.g., test kits) used in detecting the presence of hscdc6 in a sample comprising an antibody or functional portion thereof which binds to hscdc6, and one or more ancillary reagents suitable for detecting the presence of a complex between the antibody or portion thereof and said hscdc6. In particular, the antibodies can be hCdc6-26, hCdc6-37, hCdc6-34, hCdc6-39, or hCdc6-41. The kit can also further comprise one or more reagents for detecting an additional marker for a proliferative disorder (e.g., a marker that is not hscdc6, such as a Mcm protein).